Novel methods for exploration and engineering of regulatory ncRNA in bacteria

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Amador, Paul

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The need for sustainable resources has spurred the establishment of microorganisms as platforms of chemical production. These “microbial factories” are engineered to maximize production of valuable chemicals. Essential to the engineering of these production strains, is the ability to control and regulate gene expression. The engineering of native and synthetic pathways of production relies on the ability to control the levels of intermediate and end products, as well as the mechanisms that impose control and induce gene expression. Traditionally, this involves the targeted deletion and overexpression of specific genes of interest within the genome. Of late, however, there has been interest in exploring the regulatory capacity of RNA for biotechnology. Discovery of regulatory elements, such as riboswitches and sponge RNAs, has advanced the capacity and utility of RNA for bioengineering. In my work, I have developed an in vivo screen for detecting RNA elements that are responsive to stress in the radiation-resistant bacteria, Deinococcus radiodurans. Investigations of the response and regulatory capacity of ncRNA, specifically 5’ UTRs, are especially valuable in this organism as radiation induces stress similar to that of oxidation and aging. Notably, this work has yielded the discovery of an mRNA-based regulon, previously thought to act only at the promoter level to regulate multiple genes associated with radiation and desiccation response. The expanded RNA-based model I present for this regulon serves to validate the advantages of RNA-level regulation for rapid and robust responses that make such RNA regulatory elements valuable for biotechnological applications. Moreover, my research into RNA regulation involved the characterization and engineering of the regulatory ncRNA, csrB, that, in concert with the regulatory protein CsrA, controls the expression of hundreds of genes throughout gamma proteobacteria. Specifically, I utilized an in vivo assay to generate an accessibility profile for csrB (for which little structural information exists in E.coli) to determine sites that are likely binding the regulatory protein, CsrA. This accessibility profile informed the rational engineering of csrB to alter affinity for binding CsrA dimers and ultimately “tune” the regulatory capacity of the Csr global regulatory system for producing complex phenotypes desirable for “microbial factories”.



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